A mechanism of controlling gene expression mediated by a small RNA having a length of about 20 to 30 nucleotides is called gene silencing. This mechanism is widely reserved in a variety of biological species from yeast to human and has been revealed to be involved in regulation of many important vital phenomena such as development, metabolism, and virus infection control. In addition, at present, RNA interference (RNAi) method, which inhibits gene expression through a gene silencing mechanism using small RNA, has been establishing a position as an indispensable gene functional analysis tool in the biological research field. Furthermore, such small RNA shows the effect of suppressing the expression of, for example, a gene or virus in vivo and is therefore expected as a next-generation nucleic acid drug.
One example of the small RNA mediating gene silencing is a small interfering RNA (siRNA), which is generated by Dicer enzyme processing of, for example, a foreign long double-stranded RNA or an RNA with a hairpin structure. One strand (called antisense strand) of the generated double-stranded siRNA is incorporated into a complex, called RNA-induced silencing complex (RISC), binds to a target mRNA having a complementary sequence, and induces cleavage of the target mRNA to sequence-specifically suppress expression of the gene. Meanwhile, a group of functional small RNAs, called microRNAs (miRNAs), is present in a cell. The miRNA is an endogenous small RNA that is natively expressed in the cell and is believed to suppress expression of another gene. The miRNA is generated by Drosha and then Dicer enzyme processing of a long miRNA precursor having a hairpin structure. It is believed that one strand of the generated double-stranded miRNA is, as in the case of a siRNA, incorporated into an RISC and then binds to an mRNA having a 3′-untranslated region including a sequence partially complementary to the miRNA to inhibit translation into a protein (see Non Patent Literature 1 for the review).
The small double-stranded RNA that mediates gene silencing is generated by a Dicer enzyme, as described above, and therefore has a constant structure, i.e., being composed of two RNAs each consisting of about 21 to about 24 nucleotides and forms a double-stranded RNA having an overhang of two nucleotides at the 3′-end of each strand. Accordingly, many of the siRNAs that are generally used in the present RNAi method are chemically synthesized products having an overhang of two nucleotides at each 3′-end. There have been some studies on the correlation between the structure and the activity of such siRNAs. For example, Elbashir et al. tested the activities of siRNA molecules each having a length of 21 nucleotides and having overhangs of various lengths at the 3′- or 5′-ends and confirmed that an overhang of two nucleotides at a 3′-end shows the highest efficiency (Non Patent Literature 2). In addition, they have reported that although the RNAi activity disappears by completely replacing one strand or both strands of the siRNA with 2′-deoxyribonucleotides, similar replacement of only the 3′-overhang of two nucleotides does not affect the RNAi activity (Non Patent Literature 2). Sun et al. showed that an asymmetric double-stranded RNA (aiRNA) of which the sense strand is shorter than a typical siRNA with a length of 21 nucleotides has an effective RNAi activity and reduces the off-target effect of the sense strand (Non Patent Literature 3). Bramsen et al. showed a design of a novel siRNA (sisiRNA) composed of an intact antisense strand and two short (a length of 10 to 12 nucleotides) sense strands (Non Patent Literature 4). The sisiRNA has been reported to have an activity equivalent to that of usual siRNA and to be low in off-target effect. These siRNA molecules having novel structures may have modifications, such as locked nucleic acid (LNA), and are further expensive than usual expensive RNAs having high synthetic cost by its nature. Thus, they have difficulty in practical use. Furthermore, the effect of controlling gene expression of siRNA varies considerably depending on the sequence design, and the effect is insufficient in many cases. In such a case, an increase in dose of the siRNA is required, which leads to a disadvantage that a risk of undesirable effects such as an off-target effect is raised.